Reverse transcription is the process by which a retrovirus such as HIV-1 converts its single-stranded (ss) RNA genome into a double-stranded DNA copy that is integrated into host chromosomal DNA. This process is complex and is catalyzed by the virion-associated enzyme, reverse transcriptase (RT). However, another viral protein, the nucleocapsid protein (NC), is also required for productive viral DNA synthesis. (A) We study the mechanistic basis for NC activity. HIV-1 NC is a small (7 kDa), basic nucleic acid binding protein with two zinc fingers (ZFs), each containing the invariant CCHC zinc-coordinating motifs. It is also a nucleic acid chaperone, i.e., NC facilitates remodeling of nucleic acid structures so that the most thermodynamically stable conformations are formed. This property is critical for promoting efficient and specific reverse transcription. (i) Recent studies have focused on Gag C-terminal cleavage products i.e., NCp15 (NCp9-p6), NCp9 (NCp7-SP2), and mature NC (NCp7), and comparison of their nucleic acid chaperone activities in reconstituted systems modeling early reverse transcription events. Assays of tRNALys3-primed (-) strong-stop (SS) DNA synthesis show that while NCp9 and NCp7 exhibit comparable activity, NCp15 makes 2-fold less product. Interestingly, synthesis of (-) SSDNA is dramatically reduced when the NCp9 concentration is increased. Similarly, although NCp9 is more active than NCp7 or NCp15 in minus-strand transfer reactions, at high concentrations of NCp9, synthesis of the transfer product reaches a plateau and then decreases as incubation proceeds. Results of aggregation assays suggest that this unusual behavior reflects NCp9's strong nucleic acid aggregation activity, which is enhanced by its highly basic C-terminal SP2 domain. Interestingly, the SP2 peptide by itself can stimulate the annealing reaction in minus-strand transfer. NCp15 has much lower minus-strand transfer activity than NCp9 or NCp7, which we hypothesized is due to NCp15's acidic C-terminal p6 domain. Indeed, mutants with Ala substitutions in the acidic residues have improved chaperone activity and considerably lower Kd values for binding single-stranded (ss) DNA. Taken together, these results are in good agreement with the biological activity of virions with mutations in protease cleavage sites: (i) HIV-1 containing NCp15 is not infectious; and (ii) while virus containing NCp9 is initially infectious, continued passage in culture results in reversion to wild type (Coren et al. J. Virol. 81:10047-10054, 2007; De Marco et al. J. Virol. 86:13708-13716, 2012). Thus, our data help to explain why fully processed NCp7 has evolved as the critical cofactor in HIV-1 reverse transcription and is optimal for viral fitness. (ii) During synthesis of (-) SSDNA, the RNase H activity of RT cleaves the viral RNA template, generating small 5-prime terminal RNA fragments that remain annealed to the DNA. Unless these fragments are removed, the minus-strand transfer reaction, required for (-) SSDNA elongation, cannot proceed. We hypothesized that fragment removal could be facilitated by NC destabilization of the short duplex and/or by RNase H cleavage. To test this hypothesis, we used an NC-dependent system that models minus-strand transfer. We showed that that the presence of short terminal fragments pre-annealed to (-) SSDNA has no impact on strand transfer, implying efficient fragment removal. Moreover, in reactions with an RNase H-minus RT mutant, NC alone is able to facilitate fragment removal, albeit less efficiently than in the presence of both RNase H and NC. Results obtained from novel gel-mobility shift and FRET assays, which each directly measure RNA fragment release from a duplex in the absence of DNA synthesis, demonstrated that the architectural integrity of NC's ZF domains is absolutely required for this reaction. These findings are in excellent agreement with our earlier studies of the tRNA removal step in plus-strand transfer (Wu et al. J.Virol. 73:4794-4805, 1999) and the ability of NC to block mispriming during initiation of plus-strand DNA synthesis (Post et al. NAR 37:1755-1766, 2009). Thus, HIV-1 uses a common mechanism for the RNA removal reactions required for successful reverse transcription. (B) Our interest in host proteins that affect HIV-1 reverse transcription and replication has led us to investigate the activities of human APOBEC3 (A3) proteins. We have been studying human A3A, which like other A3 family members, is a cytidine deaminase that converts deoxycytidine residues to deoxyuridine in single-stranded (ss) DNA and functions as a DNA mutator. A3A inhibits a wide range of viruses, including HIV-1 and other retroviruses, and also displays potent activity against endogenous retroelements such as LINE-1. Our collaborators, who are structural biologists, have determined the solution structure of A3A at high resolution using NMR spectroscopy and a paper describing our initial studies of the structure and biochemical properties of A3A was published recently in Nature Communications (Byeon et al. Nature Comm. 4:1890, 2013). In continuing work on A3A, we have taken advantage of the new information to perform structure-guided mutagenesis studies designed to characterize A3A's enzymatic, nucleic acid binding, and biological activities. We show that A3A binds and deaminates ssDNA in a length-dependent manner. Surprisingly, although A3A also binds ssRNA, NMR analysis demonstrates that the RNA and DNA binding interfaces differ. Moreover, no deamination of ssRNA is detected in real-time NMR assays. In experiments on LINE-1 retrotransposition, assays with active- and non-active site A3A mutants reveal that the absence of deaminase activity per se does not always result in a loss of anti-LINE-1 activity, demonstrating that these two activities are not linked. We have also performed experiments that indicate a mechanism for A3A's recently reported ability to mutate normally double-stranded genomic DNA, an activity that is implicated in carcinogenesis. Taken together, our studies provide new insights into the molecular properties of A3A and its role in multiple cellular and antiviral functions. Current work is also focused on probing the determinants of A3H cytidine deaminase activity, using site-directed sequence- and structure-guided mutagenesis (based on a homology model) in conjunction with biochemical and biological assays. (C) Our laboratory has demonstrated that the short flexible peptide, known as the interdomain linker, which connects the N- and C-terminal domains of CA, is a critical determinant of proper core assembly and stability. In the course of this work, we identified two novel mutants (P147L and S149A). These mutants, while poorly infectious, display an attenuated phenotype and surprisingly, their infectivity is rescued when env-minus virions are pseudotyped with the vesicular stomatitis envelope glycoprotein. Moreover, despite having unstable cores, these mutants (i) synthesize viral DNA in infected cells, although less efficiently than WT virus, (ii) assemble a significant number of viral cores with seemingly normal architecture, and (iii) assemble tubular structures in vitro that resemble WT assemblies. In future work, we plan to examine in vitro CA assemblies by cryo-electron tomography to determine the nature of the defect in the cores of these unusual mutants.